One-electron reduction of selenomethionine oxide

Experimental evidence is provided that selenomethionine oxide (MetSeO) is more readily reducible than its sulfur analogue, methionine sulfoxide (MetSO). Pulse radiolysis experiments reveal an efficient reaction of MetSeO with one-electron reductants, such as e^-_aq (k = 1.2 × 10¹⁰M^-1s^-1), CO^·-₂ (k = 5.9 × 10⁸ M^-1s^-1) and (CH₃)₂) C^·OH (k = 3.5 × 10⁷M^-1s^-1), forming an intermediate selenium-nitrogen coupled zwitterionic radical with the positive charge at an intramolecularly formed Se_∴ N 2σ/1σ* three-electron bond, which is characterized by an optical absorption with λ_max at 375 nm, and a half-life of about 70 μs. The same transient is generated upon HO^· radical-induced one-electron oxidation of selenomethionine (MetSe). This radical thus constitutes the redox intermediate between the two oxidation states, MetSeO and MetSe. Time-resolved optical data further indicate sulfur-selenium interactions between the Se_∴ N transient and GSH. The Se_∴ N transient appears to play a key role in the reduction of selenomethionine oxide by glutathione.     

Mechanism of reaction of nitrogen dioxide radical with hydroxycinnamic acid derivatives: A pulse radiolysis study

Nitrogen dioxide radical (NO^·₂) is known as a toxic agent produced in the metabolism of nitrates and nitrites. By the use of the pulse radiolysis technique, the mechanism of the reaction of NO^·₂ radical with hydroxycinnamic acid derivatives (HCA) was studied and the rate constants have been measured. The rate constants were found to be 7.4 × 10⁸, 7.2 × 10⁸, 8.6 × 10⁸ dm³ mol^-1s^-1 for ferulic acid, sinapic acid and caffeic acid, respectively. The reactions produce the corresponding phenoxyl radical.     

Interactions of superoxide anion with enzyme radicals: kinetics of reaction with lysozyme tryptophan radicals and corresponding effects on tyrosine electron transfer

The kinetics of O^·-₂ reaction with semi-oxidized tryptophan radicals in lysozyme, Trp^·(Lyz) have been investigated at various pHs and conformational states by pulse radiolysis. The Trp^·(Lyz) radicals were formed by Br^·-₂ oxidation of the 3-4 exposed Trp residues in the protein. At pH lower than 6.2, the apparent bimolecular rate is about 2 × 10⁸M^-1s^-1; but drops to 8 × 10⁷M^-1s^-1 or less above pH 6.3 and in CTAC micelles. Similarly, the apparent bimolecular rate constant for the intermolecular Trp^·(Lyz) + Trp^·(Lyz) recombination reaction is about (4-7 × 10⁶M^-1s^-1) at/or below pH 6.2 then drops to 1.3-1.6 × 10⁶M^-1s^-1 at higher pH or in micelles. This behavior suggests important conformational and/or microenvironmental rearrangement with pH, leading to less accessible semioxidized Trp^· residues upon Br^·-₂ reaction. The kinetics of Trp^·(Lyz) with ascorbate, a reducing species rather larger than O^·-₂ have been measured for comparison. The well-established long range intramolecular electron transfer from Tyr residues to Trp radicals-leading to the repair of the semi-oxidized Trp^·(Lyz) and formation of the tyrosyl phenoxyl radical is inhibited by the Trp^·(Lyz)+O^·-₂ reaction, as is most of the Trp^·(Lyz)+Trp^·(Lyz) reaction. However, the kinetic behavior of Trp^·(Lyz) suggests that not all oxidized Trp residues are involved in the intermolecular recombination or reaction with O^·-₂. As the kinetics are found to be quite pH sensitive, this study demonstrates the effect of the protein conformation on O^·-₂ reactivity. To our knowledge, this is the first report on the kinetics of a protein-O^·-₂ reaction not involving the detection of change in the redox state of a prosthetic group to probe the reactivity of the superoxide anion.     

Scavenging of reactive oxygen species by chlorophyllin: An ESR study

The antioxidant effects of chlorophyllin (CHL), a water-soluble analog of the green plant pigment chlorophyll, on different reactive oxygen species (ROS) were investigated by electron spin resonance (ESR) spectroscopy. As a standard, we have used the ability of CHL to scavenge the stable 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical. CHL inhibits the formation of 5,5-dimethyl-1-pyrroline-N-oxide adduct with hydroxyl radical (DMPO-OH adduct) generated by γ-radiation in a dose-dependent manner. At a concentration of 1 mM, CHL caused more than 90% inhibition of ESR signal intensity of this adduct. However, the results obtained with the Fenton reaction were different. We also found evidence for the inhibition of ¹O₂-dependent formation of the 2,2,6,6-tetramethyl-piperidine oxide (TEMPO) radical during photosensitization of methylene blue with visible light. CHL was also able to inhibit hydrogen peroxide induced oxidation of phenol red. The rate constant of the reaction of CHL with H₂O₂ was found to be 2.7×10⁶ M^-1s^-1. In conclusion, CHL has potent antioxidant ability involving scavenging of various physiologically important ROS.     

The Tetrazolium Dyes MTS and XTT Provide New Quantitative Assays for Superoxide and Superoxide Dismutase

The tetrazolium dyes MTS and XTT were reduced to their soluble formazans by superoxide radical anions (O₂^_) produced by the oxidation of xanthine by xanthine oxidase under standard conditions. These reactions were compared to the well-known reductions of NBT and cytochrome c by the xanthine/xanthine oxidase system. Reduction of the dyes was completely inhibited by superoxide dismutase (SOD). Rate constants for the reaction of MTS and XTT with O₂^_: were estimated at 1.3 × .1 × 10⁵ M-¹s^-1 and 8.6 × .8 × 10⁴ M^-1s^-1 respectively. The stable MTS and XTT formazans have high extinction coefficients in the visible range which enable sensitive detection and quantification of superoxide radicals, avoiding some of the problems inherent in assays based on production of the insoluble NBT formazan. MTS and XTT have considerable potential both for the quantitative assay of radical production in living tissues and for the assay of superoxide dismutase activity in tissue extracts. Implications for the interpretation of cell culture growth assays which employ these dyes are discussed.     

Oxygen-copper (II) interplay in the repair of semi-oxidized urate by quercetin bound to human serum albumin

The 1:1 complex of copper (II) and human serum albumin (HSA) slowly reacts with radiolytically generated ^•O₂^- radical-anion at a rate constant of 6.1×10₆ M_-1 s_-1. Absorbance and fluorescence spectroscopies demonstrate that addition of an equimolar portion of quercetin (QH₂) to the solution of the copper (II)-HSA complex induces a relocalization of the copper resulting in a ternary copper (II)-QH₂-HSA complex. This form of quercetin slowly oxidizes in air-saturated solutions. A 10-fold excess urate, a plasma antioxidant, cannot displace copper (II) bound to HSA. In N₂O-saturated solutions the ternary complex form of QH₂ can repair the urate radical with a rate constant of 2.7×10₆ M_-1 s_-1 by an electron transfer reaction similar to that observed in the absence of copper (II). In O₂-saturated solutions and in the absence of copper, HSA-bound QH₂ fails to repair the urate radical because of the fast competitive reaction of ^•O₂^- with urate radicals. However, addition of equimolar copper (II) restores the electron transfer from QH₂ to the urate radical. These contrasting results are tentatively explained either by an enhanced reactivity of copper (II) with ^•O₂^- in the ternary complex or by direct production of quercetin radicals via a copper-catalyzed reduction of the ^•O₂^- radicals by QH₂.     

On the Reaction of Superoxide With DMPO/*OOH

A kinetic model has been used to estimate the rate constant for the reaction of superoxide (O₂/OOH) with the superoxide spin adduct of 5.5-dimethylpyrroline-N-oxide. DMPO/OOH. This rate constant is estimated to be 4.9 (± 2.2) × 10⁶ M^-1 s^-1, pH 7.4 and 25°C.     

Catalysis of plastocyanin electron self-exchange by redox-inert multivalent cations

Electron self-exchange in solutions of the ‘blue’ copper protein plastocyanin is catalysed by the redox-inert multivalent cations Mg²⁺ or Co(NH₃)³⁺₆. Measurements of specific ¹H-NMR line broadening with 50% reduced solutions in the presence of these cations show that electron exchange proceeds through encounters of cation-protein complexes which dissociate at high ionic strength. In the presence of 8mM (5 equivalents/total protein) Co(NH₃)³⁺₆, with 10 mM cacodylate (pH^*6.0) as background electrolyte, the bimolecular rate constant at 25°C is 7 × 10⁴ M⁻¹·s⁻¹. For comparison, the ‘electrostatically screened’ rate constant measured in 0.1 M KCl in the absence of added multivalent cations is ˜ 4 × 10³ M¹·s⁻¹.

Plastocyanin Electron self-exchange NMR Protein-protein interaction Multivalent cation Blue copper protein     

Kinetics of the reactions of nitrogen dioxide with glutathione,cysteine, and uric acid at physiological pH

Nitrogen dioxide (NO₂^•) is a key biological oxidant. It can be derived from peroxynitrite via the interaction of nitric oxide with superoxide, from nitrite with peroxidases, or from autoxidation of nitric oxide. In this study, submicromolar concentrations of NO₂^• were generated in < 1 μs using pulse radiolysis, and the kinetics of scavenging NO₂^• by glutathione, cysteine, or uric acid were monitored by spectrophotometry. The formation of the urate radical was observed directly, while the production of the oxidizing radical obtained on reaction of NO₂^• with the thiols (the thiyl radical) was monitored via oxidation of 2,2′-azino-bis-(3-ethylthiazoline-6-sulfonic acid). At pH 7.4, rate constants for reaction of NO₂^• with glutathione, cysteine, and urate were estimated as 2 × 10⁷, 5 × 10⁷, and 2 × 10⁷ M⁻¹ s⁻¹, respectively. The variation of these rate constants with pH indicated that thiolate reacted much faster than undissociated thiol. The dissociation of urate also accelerated reaction with NO₂^• at pH > 8. The thiyl radical from GSH reacted with urate with a rate constant of 3 × 10⁷ M⁻¹ s⁻¹. The implications of these values are: (i) the lifetime of NO₂^• in cytosol is < 10 μs; (ii) thiols are the dominant ‘sink’ for NO₂^• in cells/tissue, whereas urate is also a major scavenger in plasma; (iii) the diffusion distance of NO₂^• is 0.2 μm in the cytoplasm and < 0.8 μm in plasma; (iv) urate protects GSH against depletion on oxidative challenge from NO₂^•; and (v) reactions between NO₂^• and thiols/urate severely limit the likelihood of reaction of NO₂^• with NO• to form N₂O₃ in the cytoplasm.     

Topical delivery of retinyl ascorbate: 4. Comparative anti-oxidant activity towards DPPH

The free radical scavenging properties of retinyl ascorbate (RA-AsA) were determined by monitoring the decomposition of 2,2-diphenyl-1-picrylhydrazyl (DPPH) as a function of time and in comparison with ascorbic acid (AsA), ascorbic acid palmitate (AsA-Pal), retinoic acid (RA), retinol (ROL) and retinol palmitate (Rol-Pal). The rate constant of RA-AsA (mean₃±SD) was 4.9±0.3 M^-1 s^-1, and indicated greater potency as an antioxidant compared to the rest of the test compounds (AsA 3.4±0.4 M^-1 s^-1, AsA-Pal, 2.9±0.2 M^-1 s^-1, RA 1.4±0.3 M^-1 s^-1, ROL 1.3±0.1 M^-1 s^-1, Rol-Pal exhibited insignificant activity). The decomposition rate constant of DPPH, 5±0.6 × 10^-8 M^-1 s^-1, in ethanol and BHA, 154±3 M^-1 s^-1 were both used as control. The compound RA-2-carboxy-2-hydroxy-ethanoate was isolated by prep-TLC and was identified, by ¹³C and ¹HNMR spectroscopy, as the major by-product from the reaction of RA-AsA with DPPH, which was also found to be potent antioxidant, 2.1±0.2 M^-1 s^-1. This suggests that oxidation of AsA moiety did not lead to the production of erythrulose species, which could cause deleterious modifications of cellular proteins.     

On the Specificity of Allopurinol and Oxypurinol as Inhibitors of Xanthine Oxidase. A Pulse Radiolysis Determination of Rate Constants for Reaction of Allopurinol and Oxypurinol with Hydroxyl Radicals

Allopurinol has been employed as a “specific” inhihitor of xanthine oxidase in studies of hypoxic/ reoxygenation injury. Pulse radiolysis was used to establish rate constants for the reactions of allopurinol and its major metabolite oxypurinol with hydroxyl radicals: values were (1.45 ± 0.241 × 10⁹ M^-1 s^-1 for allopurinol and (4.95 ± 0.84) × 10⁹ M^-1 s^-1 for oxypurinol. These rate constants show that, in view of the amounts of allopurinol that have been used in animal studies. hydroxyl radical scavenging by this molecule could contribute to its biological actions. especially if animals are pre-treated with allopurinol. so allowing oxypurinol to form. The ability of allopurinol to protect tissues not containing xanthine oxidase against reoxygenation injury may be related to radical scavenging by allopurinol and oxypurinol.     

Photosensitization mechanism of active species by the complex of hypocrellin B with aluminum ion

To improve the water solubility and red absorption of the parent hypocrellin B (HB), the complex of HB with aluminum ion has been first synthesized in high yield. The photodynamic action of Al³⁺-HB, especially the generation mechanism of active species, ([Al³⁺-HB]^·-, O^·-₂ and ¹O₂) was studied using electron paramagnetic resonance (EPR) and spectrophotometric methods. In the deoxygenated DMSO solution of Al³⁺-HB, the semiquinone anion radical of Al³⁺-HB is photogenerated via the self-electron transfer between the excited and ground state species. The presence of electron donor significantly promotes the reduction of Al³⁺-HB. When oxygen is present, superoxide anion radical (O^·-₂) is formed via the electron transfer from [Al³⁺-HB]^·- to the ground state molecular oxygen. Singlet oxygen (¹O₂) can be produced via the energy transfer from triplet Al³⁺-HB to ground state oxygen molecules. Furthermore, it is very significant that the accumulation of [Al³⁺-HB]^·- would replace that of O^·-₂ or ¹O₂ with the consumption of oxygen in the sealed system.     

4-Mercaptoimidazoles derived from the naturally occurring antioxidant ovothiols 1. Antioxidant properties

4-Mercaptoimidazoles derived from the naturally occurring family of antioxidants, the ovothiols, were assayed for their antioxidant properties. These compounds are powerful HOCI scavengers, more potent than the aliphatic thiol N-acetylcysteine. They react slowly with hydrogen peroxide with second order rate constants of 0.13-0.89 M^-1 s^-1. Scavenging of hydroxyl radical occurs at a diffusion-controlled rate (k = 2.0-5.0 × 10¹⁰ M^-1 s^-1) for the most active compounds, which are also able to inhibit copper-induced LDL peroxidation. The combination of radical scavenging and copper chelating properties may explain the inhibitory effects on LDL peroxidation. Two molecules of mercaptoimidazole can chelate a copper ion and form a square planar complex detected by EPR. Compounds bearing an electron-withdrawing group on position 2 of the imidazole ring are the most potent antioxidant molecules in this series.     

Oxidation-Reduction Reactions of Iron Bleomycin in the Absence and Presence of DNA

Using the pulse radiolysis technique, we have demonstrated that bleomycin-Fe(III) is stoichiometrically reduced by CO₂^- to bleomycin-Fe(II) with a rate of (1.9 ± 0.2) × 10⁸M^-1s^-1. In the presence of calf thymus DNA, the reduction proceeds through free bleomycin-Fe(III) and the binding constant of bleomycin-Fe(III) to DNA has been determined to be (3.8 ± 0.5) x 10⁴ M^-1. It has also been demonstrated that in the absence of DNA O₂^-1 reacts with bleomycin-Fe(III) to yield bleomycin-Fe(II)O₂, which is in rapid equilibrium with molecular oxygen, and decomposes at room temperature with a rate of (700 ± 200) s^-1. The resulting product of the decomposition reaction is Fe(III) which is bound to a modified bleomycin molecule. We have demonstrated that during the reaction of bleomycin-Fe(II) with O₂, modification or self-destruction of the drug occurs, while in the presence of DNA no destruction occurs, possibly because the reaction causes degradation of DNA.     

The Kinetics of the Reaction of Ferritin with Superoxide

Using pulse radiolysis and competition kinetics with cytochrome c, the reaction of superoxide with horse spleen ferritin was investigated. The second-order rate constant is estimated to be 2 ± 1 × 10⁶dm³mol^-1s^-1     

The antioxidant action of N-acetylcysteine: Its reaction with hydrogen peroxide, hydroxyl radical, superoxide, and hypochlorous acid

N-acetylcysteine has been widely used as an antioxidant in vivo and in vitro. Its reaction with four oxidant species has therefore been examined. N-acetylcysteine is a powerful scavenger of hypochlorous acid (H---OCl); low concentrations are able to protect ₁-antiproteinase against inactivation by HOCl. N-acetylcysteine also reacts with hydroxyl radical with a rate constant of 1.36 × 10¹⁰ M⁻¹s⁻¹, as determined by pulse radiolysis. It also reacts slowly with H₂O₂, but no reaction of N-acetylcysteine with superoxide (O₂⁻) could be detected within the limits of our assay procedures.     

The role of N-acetylcysteine in protecting synovial fluid biomolecules against radiolytically-mediated oxidative damage: A high field proton NMR study

High field proton (¹H) NMR spectroscopy has been employed to evaluate the abilities of the antioxidant thiol drug N-acetylcysteine and exogenous cysteine to protect metabolites present in intact inflammatory synovial fluid samples against oxidative damage arising from gamma-radiolysis (5.00 kGy) in the presence of atmospheric O₂. Although oxidation of urate to allantoin by radiolytically-generated *OH radical was readily circumventable by pre-treatment of synovial fluids with N-acetylcystine (1.00 or 3.00 × 10^-3 mol · dm^-3) or cysteine (1.00, 2.00 or 5.00 × 10^-3 mol · dm^-3), both thiols offered only a limited protective capacity with respect to hyaluronate depolymerisation and the production of formate from carbohydrates in general. Radiolytic products generated from the added thiols (predominantly their corresponding disulphides) were simultaneously detectable in ¹H Hahn spin-echo spectra of gamma-irradiated synovial fluids, permitting a quantitative evaluation of the radioprotective capacity of these agents. It is concluded that the multicomponent analytical ability of high field ¹H NMR spectroscopy provides much useful molecular information regarding mechanisms associated with the radioprotectant actions of thiols in intact biofluids.     

The diffusion-controlled reaction of semioxidized tryptophan with the superoxide radical anion

From pulse radiolysis measurements in oxygenated aqueous solution, the semioxidized tryptophan radical (Trp·— formed by the one-electron oxidation of Trp by Br₂^- radical—has been shown to oxidize the superoxide radical anion with a rate constant of k = 2 × 10⁹ M⁻¹ s⁻¹. Proof of this reaction is found in addition of superoxide dismutase (SOD) to the system, which totally eliminates the contribution of the Trp^· + O₂^- mechanism to Trp^· decay. Little, if any, reaction of molecular oxygen with Trp^· may be observed on the time scale of the pulse radiolysis experiment.     

Electrochemical Characteristics of Nitro-Heterocyclic Compounds of Biological Interest IV. Lifetime of the Metronidazole Radical Anion

Electrochemical studies on metronidazole using mixed aqueous/dimethylformamide (DMF) solvents have allowed us to generate the one-electron addition product, the nitro radical anion, RNO^-₂. Cyclic volt-ammetric techniques have been employed to study the tendency of RNO^-₂ to undergo further chemical reaction. The return-to-forward peak current ratio. ip/ip_f. was found to increase towards unity with increasing DMF content of the medium, indicating the extended lifetime of RNO^-₂. Second order kinetics for the decay of RNO^-₂ were established at all DMF concentrations examined. Extrapolation has allowed the rate constant and a first half-life of 8.4 × 10⁴dm²/mol-sec and 0.059 seconds respectively, to be determined for the decay of RNO^-₂ in a purely aqueous media. This is impossible by direct electrochemical measurement in water. due to a different reduction mechanism, giving the hydroxylamine derivative in a single 4-electron step. The application of the technique to other nitro-aromatic compounds is discussed.     

The reactions of oxicam and sulfoanilide non steroidal anti-inflammatory drugs with hypochlorous acid: determination of the rate constants with an assay based on the competition with para-aminobenzoic acid chlorination and identification of some oxidation products

Hypochlorous acid (HOCl) is an oxygen-derived species involved in physiological processes related to the defence of the organism that may cause adverse effects when its production is insufficiently controlled. In order to examine its reactivity with potential scavenging molecules from the non steroidal anti-inflammatory drugs (NSAIDs) family, a competition assay based on para-aminobenzoic acid (PABA) chlorination was developed. The original optimised in vitro fluorimetric procedure offered the possibility to determine rate constants (k_s) for the reaction with HOCl in physiologically relevant conditions. The specificity of the system was improved by a liquid chromatography (LC) which allows the separation of the drugs and their oxidation products. After determination of the rate constant for PABA chlorination by HOCl (mean±SD in M_-1 s_-1: 4.3±0.3×10³), the applied mathematical model for a chemical competition permits to obtain linear curves from competition studies between several NSAIDs and PABA. Their slopes provided the following rate constants for the different studied drugs: tenoxicam: 4.0±0.7×10³, piroxicam: 3.6±0.7×10³, lornoxicam: 4.3±0.7×10³, meloxicam: 1.7±0.3×10⁴, nimesulide: 2.3±0.6×10². Meloxicam therefore reacted significantly faster than the other oxicams and nimesulide, which is the weakest scavenger of the studied series. The identification of some of the oxidation products by NMR or MS permitted to explore the reaction mechanism and to examine some aspects of the structure/activity relationships for the molecules of the same chemical family.